The specific aims of this proposal are to gain a molecular understanding of how the heat shock transcription factor (HSF) is activated by heat shock, and to determine how activation of the factor occurs. We plan on focusing on the signal transduction pathway that modulates the activity of the HSF from Drosophila and yeast. We will determine the residues of HSF that are phosphorylated in normally growing cells, heat shocked cells and during recovery from heat shock. We will generate point mutations in the phosphorylated residues of the HSFs and determine whether they are regulatory in vivo. We will identify and isolate kinases present in extracts prepared from non-shocked, shocked and recovering cells and compare their phosphorylation pattern with that of HSF in vivo. Those kinases which generate a 2D phosphopeptide map pattern similar to or overlapping with the in vivo map will be purified and characterized. It is likely that these kinases play an important role in the signal transduction of the stress response. Drosophila and S. cerevisiae HSF's differ in their mode of regulation in that nuclear entry of the Drosophila factor is also regulated by stress. We have identified an important domain of the Drosophila HSF which plays a key role in this process. We propose to identify co-factors which associate with this domain in vivo, purify them, and attempt to determine their mechanism of action.